PROJECT 4 PROJECT SUMMARY Pannexin 1 (Panx1) is a widely-expressed membrane ion channel that, when activated, leads to transmembrane flux of large molecules (i.e., nucleotides, other metabolites) that can mediate intercellular signaling in multiple (patho)physiological contexts (e.g., see Projects 1-3). Thus, understanding the different cellular and molecular mechanisms for channel activation, and the determinants for large molecule permeation, are of paramount importance to reveal novel potential therapeutic strategies for pathway-specific pharmacological intervention that could selectively modulate permeation of specific signaling metabolites in different contexts. Among well-established Panx1 activation mechanisms, that mediated by G?q protein-coupled receptors (G?qPCRs) is widespread, but the essential cellular, molecular and biophysical mechanisms that mediate this prevalent form of channel activation have not been elucidated. Our preliminary data implicate the salt-inducible kinase, SIK1, a serine-threonine kinase that physically associates with Panx1, and is both necessary and sufficient for channel activation. In Aim 1, supported by additional preliminary observations, we test the hypothesis that G?qPCRs signal via non-canonical pathways involving LKB1 and RhoA-mDia-HDAC6, which converge to activate SIK1 to mediate phosphorylation and activation of Panx1. For this, we use genetic and pharmacological tools, in heterologous and native systems, to determine the relevant signaling pathways and identify critical channel phosphosites by mutational, mass spectrometric and in vitro kinase approaches. In addition, we use single channel recordings to characterize properties of partially and fully receptor-activated wild type and concatenated Panx1 constructs, examining whether channels activate in the novel stepwise fashion that we recently discovered for C-terminally cleavage-activated channels. Panx1 channels are renowned for their association with nucleotide release and dye uptake. Nonetheless, it has not been established whether these large molecules actually permeate via the channel itself, and even the ionic selectivity of Panx1 has not been established. In addition, channels activated by different mechanisms display distinct single channel properties, suggesting that they may also yield distinct permeation properties that support release of specific signaling molecules. In Aim 2, we implement a proteoliposome system incorporating purified Panx1 to test the hypothesis that activated Panx1 provides a permeation pathway that supports release of various cellular constituents, and that flux of different molecules is influenced by distinct modes of channel activation. By directly measuring permeation of specific signaling metabolites through these purified Panx1 channels, we will identify the range of metabolites that can transit the channel when activated by either caspase- mediated C-terminal cleavage or SIK1-mediated phosphorylation. This work defines molecular mechanisms underlying physiologically relevant forms of Panx1 regulation, and identifies permeation properties and signaling metabolites supported by specific activation mechanisms.